Ligation to be transformed in a bit. (Issue with the vector not being cut efficiently=failed ligations?) I am cutting the original XylE midi as well as PSB1C3 containing B0014 and ligating them today (hopefully) the vector is purified, waiting on the gel run and then extraction of XylE insert. A ligation ratio must be determined from a gel analysis then dephosphorylation of the vector and then ligation.

+

*Ligation to be transformed in a bit. (Issue with the vector not being cut efficiently=failed ligations?)

-

Florian is performing a mini prep on pVeg promoter which if it is correctly identified in the gel i can use to ligate to my XylE + PSB1C3-B0014.

+

*I am cutting the original XylE midi as well as PSB1C3 containing B0014 and ligating them today (hopefully) the vector is purified, waiting on the gel run and then extraction of XylE insert. A ligation ratio must be determined from a gel analysis then dephosphorylation of the vector and then ligation. This will produce a promoter-less XylE+terminator vector which will be readily available to switch in the desired promoter (E+S cut)

-

Piotr is PCRing the reverse version of XylE that will be under the control of inducible promoter LacI (not delivered from synthesis yet) that will become a testing construct.

+

+

*Florian is performing a mini prep on pVeg promoter which if it is correctly identified in the gel i can use to ligate to my XylE + PSB1C3-B0014.

+

*Piotr is PCRing the reverse version of XylE that will be under the control of inducible promoter LacI (not delivered from synthesis yet) that will become a testing construct.

Revision as of 10:19, 21 September 2010

XylE team Lab Objectives

Testing expression of XylE in E.coli and characterization under the control of a constitutive promoter

Construction of the -ComE promoter/XylE fussion protein- expression system

Construction of the -LacI promoter/XylE fusion construct- expression system

Lab notes and schedule

Week 6

Week 6

Monday

Tuesday

Wednesday

Thursday

Friday

Morning

mini-prep kit of XylE-transformed E.coli (already overnight grown)

Lunch Break

Afternoon

Starting of “Testing expression of XylE in E.coli” objective

1)Annealing EcoRI and speI oligos to J23101 promoter which will be annealed later in front of the RBS-XylE registry gene (overnight)

gel analysis of mini-prep derived XylE plasmid.(requires first digestion of the vector with restriction enzymes)

Thursday, 12-Aug-2010

anealing DNA strands of J23101 promoter in a waterbath

we constructed the standard E.coli promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assemply to construct a promoter-RBS-XylE design in a psB1C3 vector. E.coli will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted biobricks.

prepared two overnight cultures of XylE transormed E.coli (one 50microliters and one of 450 microliters)

these cultures are going to be used tomorrow for mini-prepping. Miniprep will allow us to isolate E.coli's plasmid DNA(which contains the XylE gene).

Friday, 13-Aug-2010

mini-prep of XylE transformed E.coli

Mini-prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasnid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini-prep will later be used for the midi-prep (that gives out higher yeilds of DNA needed for cloning).

gel analysis of plasmid DNA retreived from mini-prep of XylE transformed E.coli, cut with restriction enzymes. From light to the left, 50micrograms digested DNA : 50 undigested DNA : 450 digested DNA : 450 undigested DNA. In lanes 1 and 3 the smaller band has a size of about 1kB which corresponds to RBS-XylE gene. The bigger bands are the cut vectors. In lanes 2 and 4 is the uncut biobrick from the registry. It appears smaller on the gel than it actually is as circular DNA travels faster through the pores of agarose gel rather than linearised DNA.

Week 7

Week 7

Monday

Tuesday

Wednesday

Thursday

Friday

Morning

midi-prep XylE E.coli (2hrs)

gel extraction kit on XylE gene trapped in agarose

3A assemply: make replica plates (overnight)

Catechol assay of E.coli

Mini-prep XylE E.coli

Midi-prep XylE E.coli

Lunch Break

Afternoon

restriction digestion of XylE for 3A assemply

gel purification of XylE from rstriction digestion

3A assemply of vector, XylE and J23101 promoter

above construct transformed in E.coli

*primers arrived!

PCR extension of XylE and GFP, round 1

Monday, 16-Aug-2010

midi-preped the XylE-transformed E.coli. The DNA yeild from the midi-prep was 134micrograms as determined by spectrophotometry. This is the XylE that is going to be used for all further experiments.

restriction digestion of midi-prepped XylE by Xbal and PstI to prepare it for 3A assemply. (with J23101 promoter and PSB1C3 vector)

Midi-prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).

I made an overnight culture of Bacillus

Tuesday, 17-Aug-2010

using the Gel Extraction Kit, we isolated the restriction enzyme cut XylE gene from the agarose gel lamp.

transformation of XL-1Blue cells with J23101 in J62001 vector from the registry. One more attemp to construct a successful promoter-xylE ligation, since we believe that the strand annealed promoter was of bad quality

Gel purification of XylE lost DNA along the way. Thus PCR XylE1 with gradient for temperature scanning (taq): PCR round 1 included 62C and only rev primer to create pool of successful extensions with with rev primer (60-62). PCR round 2 with Fwd primer and temperature scale (68-68, 72-74).

Wednesday, 25th-Aug

Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appauling(explanation for Nick:really bad) results, 2nd gel run was successful.

Performed a ligation reaction between the vector containing J23101, and XylE(one on bench and one overnight one).

Transformation of the new plasmid into competent E.coli. Successfully transformed colonies can be selected for by loss of RFP expression.

XylE-1 PCR with temperature cascade. Gel analysis and purification.

Thursday, 26th-Aug

white colonies from the promoter-XylE transformed E.coli were picked and transferred to new amp plates. One is the replica plate and the other is the catechol assay plate.

XylE-1, two rounds of PCR/purification were run to obtain a sufficiently clear band. An additional PCR run for XylE-1 was discarded afterwards.

Friday, 27th-Aug

XylE-2 PCR and gel-purification cycles (2x) to obtain clear band. XylE-2 is now ready for assembly of the GFP-XylE fusion protein.

Saturday, 28th-Aug

Preparing the annealing step between the GFP-2 and XylE-2 constructs, we discovered sequence dissimilarities in the TEV-cleavable regions which we planned to use for the annealing step. Nevertheless a PCR was run with appropriate conditions (allowing for a minimal amount of unspecific annealing).

Sunday, 29th-Aug

Gel analysis of the attempted annealing reaction of GFP-2 XylE-2 showed unsufficiently clear bands for gel-purification. A new reaction is being prepared: 10 rounds of annealing PCR, followed by addition of primers (5' primer for GFP-2 and 3' primer for XylE-2) in order to introduce an amplification step in the reaction. --- Following Kirstins advice, we are discarding this reaction and wait for the arrival of new primers for XylE-2 (5' + TEV) and GFP-2 (3' +TEV).

Week 9

Week 9

Monday

Tuesday

Wednesday

Thursday

Friday

Morning

mini-prep of promoter-XylE transformed E.col

design of reverse primer for GFP to add the corrected TEV sequence to the construct.

Monday, 30th-Aug

Set up an overnight culture for midi using colony 24 (gel analysis showed similar results colony 24 was picked randomly.)

A new primer was designed in order to add a corrected TEV-protease-cleavable sequence to the His-GFP-Flag construct. This was controlled and ordered.

Tuesday, 31st-Aug

Midi prep of colony 24 for XylE-J23101 the final concentration was ~100ng/ul which wasnt so great but Chris says the protocol produces very poor yields.

We are performing the next building step of our vector. PSB1C3 containing terminator B0014 was cut with EcoRI and XbaI. The insert was cut with EcoRI and SpeI and both were incubated for 1.5hrs. Wolf is now running a gel to purify out the insert via gel purification and perform a PCR purification on the vector.

Advisors have decided it's best not to use Jeremy's tagged XylE due to the 93% difference. Kirsten will be tagging the registry XylE and we shall purify and assay with that instead.

We shall see purification expert Kieran tomorrow and talk through the process. Chris will also talk us through our characterization of XylE experiments- we will use the robot after it's been programmed but until then we can use the plate-reader.

Thursday, 2nd-Sept

Spectra of XylE transformed E.coli after addition of catechol assay. The broad peak around 380nm wavelength arises is due to the presence of the product of the enzymatic reaction involving pyrocatechol and XylE enzyme. This peak if absent if a culture of XylE transformed cells are measured without the addition of catechol

Spectrophotometry experiments with XylE transformed E.coli in LB medium (M9 culture was contaminated) reveiled the followings: On catechol assay of the trasformed cells, the positive yellow output can be quantitively measured by a broad peak at 380nm.

Monday, 6th-Sept

PCR extension of Pveg promoter: EcoRI---Pveg---RBS-SpeI

I performed a catechol assay on the picked transformed colonies to deduce which ones were successfully transformed with the insert plus vector. 1-5 7 and 10 failed to turn yellow (1-5 were background controls)leaving 8 yellow colonies.

I had to perform a colony PCR on two selected colonies 8 and 14 to check the correct insert+terminator was present.

Annealing PCR reaction included 2 samples without and one with additional primers (XbaI-His-GFP Fwd, SpeI-XylE Rev). While the primer-including reaction did not show any clearly identifiable bands, the others showed clear, if very weak bands at 800 and 1000bp, which represent the GFP and XylE constructs respectively. No band was identifiable at the 1.7 kb range, which would have indicated a successful annealing reaction. However, problems with the lense of the gel-analyser were only discovered later to have severely reduced the band brightness. Potentially a PCR-reaction with appropriate primers to amplify an annealed product(XbaI-His-GFP Fwd, SpeI-XylE Rev), could have been successful. However the reaction mixture was disposed of before this became clear.

Wednesday, 8th Sep

A second PCR extension of Pveg promoter to introduce the RBS and cut sites. It was also gel purified and stored in gel lumbs in the freezer. (maybe needed later so keep in mind).

Overnight restriction digestion completed. Then, it was run on the gel to check if it worked and then gel purified again.

Vector PSBI-C3 was digested to remove the terminator and make it sticky for the insert (Pveg-RBS). Then it was run on the gel to check if restriction has worked, but the gel didn't run far enough to determine easily between undigested and digested vector.

Thursday, 9th Sep

Gel with digested and undigested vector PSBI-C3 was run and then the digested vector was purified.

Gel was run to determine the DNA concentration ratio for the ligation of PSBI-C3 and Pveg-RBS.

Vector PSBI-C3 was dephosphorylated.

Ligation of PSBI-C3 and Pveg-RBS has been set up overnight.

Gel-analysis and gel-purification of the XylE(2) amplification PCR product.

Thursday, 10th Sep

The transformation of E.Coli with Pveg-RBS in PSBI-C3 and PSBI-C3 by itself (to check see how successful dephosphoryaltion of PSBI-C3 was and estimate the percentage of bacteria that contain the insert) was completed

concentration of the midi prep of J23101-XylE-B0014 was determined to be ~600ng/ul (using new protocol)

Kyasha kindly digested my midi with EcoRI and SpeI and performed a gel analysis. The results show a potential additional plasmid contaminating my midi however the concentration of DNA was extremely high. NB Chris said that it could be sheared DNA from a midi prep step.

Friday, 10 sep

The transformation was a SUCCESS. 2x replica plates were made plate 1# 1-6 plate #2 6-11; colony 6 and colony 9 of plates 1 and 2 respectively were transfered into a 5ml liquid culture + 5ul CmR. These will later be turned into glycerol stocks. After the replica plates have grown up mini preps on a number of colonies shall be performed - this hopefully will eliminate the contaminating plasmid DNA. This will be followed by a midi prep.

Monday 13th september

Overnights weren't set up on sunday so they were made up alongside some assay cultures. J23101-XylE-B0014 colonies 8 and 10 of the replica plate #2 were picked. Chris also provided a replica plate containing 3k3 vector colonies. This was over a year old and he was unsure whether it was the correct plasmid or if the cells would grow up. I picked all available colonies 102 150 151 and 260 of kanamycin resistance. Lastly for assays 2x LB 2xM9 cultures were made 5ml +5ul antibiotic.

Tues 14th September

test confirming that yellow product of XylE enzmymatic reaction is leaks back from the cytosol into the solution.1C and 2C is the XylE producing cells after centrifuging and redissolving of the pellet and 1S and 2S is the supernatant after centrifuging

assay on plate reader of:

0-0.75 initial O.D. of transformed E.coli

0-1mM initial catechol concentration

The assay was carried out with E.coli, top ten spcies, transformed with J23101-XylE-B0014 in pSB1C3 vector.The overnight culture wastransfered in new medium this morning for 4 hrs before assaying. LB medium was used for dilutions and blank. Catechol was diluted in ddH2O.
Image:Catechol assay test 2.xls

Mini preps of XylE 8,10 colonies and 3K3;102 151 260 colonies. Analytical digests were performed using E+S on XylE, E+S AccI and HindIII+E for 3K3 vector respectively. The band patterns correctly identified the vector to be 3k3. Colony 10 of XylE and Colony 260 of the 3k3 vector were set up direct into 100ml LB and so will require til 12pm for midi prepping on weds. Nick performed a second plate reader assay to determine the minimal concentration of Catechol to use for assays. I took 990ul M9 culture containing colony 10 and 10ul catechol was added. This was incubated for 10 mins and then spun down. The supernatant was removed into a cuvette and the cells resuspended in M9 salts. The ODs were then read on the spectrophotometer at 380 and 600nm. It was found that control cuvette of M9 salts had a miniscule reading. M9 + cells ~0.007. Supernatant + cells were ~ 2.5 therefore we could deduce that the coloured catechol breakdown product is exported out of the cells.

Piotr:

Mini prep of 1 sample (6 samples lost due to mistake) of GFP-Xyle fusion protein and its digestion with Spe and Xba (gel to be run on the next day)

Preparing E.Coli colonies for the next day for mini prep to be redone

PCR of 6 samples of GFP-Xyle fusion

Weds 15th September

Midi preps of overnights.

In preparation for assays determining the effect of catechol and/or breakdown product on cell viability we prepared Top10 cells, one strain containing pVeg-XylE-terminator and the other containing a CMR plasmid of similar size. As the XylE cells have already been prepared, only Top10 transformation with CMR-plasmid had to be carried out.

Thurs 16th September

Top10 CMR transformation was successful. Four overnight cultures of each Top10 XylE and Top10 CMR were set off with either M9 or LB medium respectively.

Piotr: Did mini preps from bacteria with blunt ended Xyle-GFP fusion and did diagnostic using both:

- digest with XbaI and SpeI

- PCR reactions with the following primers added: HIS-GFP, XylE-Xba and the other sample with HIS-GFP and GFP-flag

The PCR reactions were not very conclusive on the gels but digests allowed to determine that colonies 1->5 seem to have the right sizes of DNA in them and on Friday they will be prepared to be sent off for sequencing

continuation of the midi from overnight step. A gel analysis was run which showed the correct bands were present. A gel purification of the protein was then performed and the bands excised.

Friday 17th September

The gel purifications of 3k3 vector and XylE were used for ligation.
3K3 was dephosphorylated and then ligated in a ratio 5:1 with the insert. This was left overnight and Chris tried to transform with it on sunday.. ligation and transformation failed :-(

Monday 20th Sept

Performed ligation again. Errors in the sequencing we received back regarding J23101-XylE-B0014.
One was a log error (in XylE) phew. The other is in PSB1C3 out of the scar site and should be OK.
Chris is currently assembling the pVEG promoter+ RBS in a vector. Once he has finished this, I will combine XylE-GFP and XylE with this promoter for comparison characterization with J23101 (in E.coli and Bacillus)

Tues 21st Sept

Ligation to be transformed in a bit. (Issue with the vector not being cut efficiently=failed ligations?)

I am cutting the original XylE midi as well as PSB1C3 containing B0014 and ligating them today (hopefully) the vector is purified, waiting on the gel run and then extraction of XylE insert. A ligation ratio must be determined from a gel analysis then dephosphorylation of the vector and then ligation. This will produce a promoter-less XylE+terminator vector which will be readily available to switch in the desired promoter (E+S cut)

Florian is performing a mini prep on pVeg promoter which if it is correctly identified in the gel i can use to ligate to my XylE + PSB1C3-B0014.

Piotr is PCRing the reverse version of XylE that will be under the control of inducible promoter LacI (not delivered from synthesis yet) that will become a testing construct.

Output Photo Gallery

First Successful Catechol Assay!

First Successful Catechol Assay!

Jeremys lawn of XylE expressing E.coli; smiley face

Catechol Assay cuvette setup

Catechol Assay cuvette setup

Determining whether catechol breakdown coloured product is in the cell or supernatant..